Volumetric analysis of epithelial morphogenesis with high spatiotemporal resolution

项目摘要 上皮片如何通过邻居的变化重塑自己以采用新的组织构象 关系和细胞形态动力学一直是发育和疾病研究中的一个关键问题。有趣的是， 在模型系统中进行的许多开创性研究在很大程度上局限于2D分析，并且 经常被挑战去想象发生在位于组织更深处的基底区域的细胞行为。 果蝇原肠胚在组织伸长过程中发生的插层运动是一个经典的 了解上皮重塑的系统，并一直是通知发育的基础 描述细胞如何在贴壁上皮中改变位置的范例。几乎所有的研究都是在 该系统仅限于对早期果蝇胚胎顶端事件的二维分析，到目前为止还没有研究。 系统地分析了驱动上皮重塑和组织延伸的全3D行为 果蝇胚胎上皮。因此，该领域剩下的最大问题之一是， 上皮细胞的体积性质影响整个细胞表面的力传播和重塑。 顶端-基底轴。关于力的来源以及力的距离和速度的根本问题 在不同的顶层-基底层的传播仍然没有答案。根据我们的初步分析，我们 已经成功地完成了第一个完整的4D果蝇嵌插上皮分割 通过使用晶格光片显微镜(LLSM)。我们发现，插层可以在任何时候引发 我们沿根尖-基底轴测量的位置。这是惊人的，因为之前的研究在很大程度上 牵涉到心尖力的产生，一项单独的研究表明，收缩力也可以起源于 从上皮的基面开始。在拟议的项目中，我们正在开发工具来执行 首次对果蝇早期胚胎的细胞嵌入进行了全面、定量的3D分析。到时候我们会的 确定驱动三维力产生的分子机制，以及不同的机械状态 存在于根尖-基底轴线上。初步数据显示高度新颖的动态肌球蛋白II和F-肌动蛋白 在侧生和基生区域表现出快速轴向繁殖的种群。它们的3D分布 正在绘制种群图，相关的肌动蛋白成核和肌球蛋白调控网络将 下定决心。这些结果将首次提供对皮质和收缩的全面理解 决定原肠上皮机械环境的网络。我们还将使用3D数据 在野生型和功能受损的背景中设置，以检查上皮力是如何传播的 使用拓扑映射度量的顶端-基础和平面尺寸。我们将确定距离有多远，在什么地方 速度，收缩力量在一个完整的，发育中的上皮中传播。这些结果将从根本上给出 回答了粘性细胞质和弹性细胞皮质如何对力驱动的位移做出反应，以及 这些位移如何在单个细胞内和跨组织传播，以驱动新的组织拓扑。

Project Summary How epithelial sheets remodel themselves to adopt new tissue conformations through changes in neighbor relationships and cell shape dynamics has been a key question in development and disease. Interestingly, many of the pioneering studies performed in model systems have largely been confined to 2D analysis, and have often been challenged to image cell behaviors that occur in basal regions that lie deeper into the tissue. The intercalation movements that occur during tissue elongation in the Drosophila gastrula have been a classic system for understanding epithelial remodeling, and have been fundamental to informing the developmental paradigms that describe how cells can change position in an adherent epithelium. Nearly all of the studies in this system have been confined to 2D analysis of apical events in the early fly embryo, and no studies to date have systematically analyzed the full 3D behaviors that drive epithelial remodeling and tissue extension in the Drosophila embryonic epithelium. Thus, one of the biggest remaining questions in the field is how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface along the entire apical-basal axis. Fundamental questions on where forces originate from as well as how far and fast forces propagate across different apical-basal layers have remained unanswered. In our preliminary analysis, we have been successful in completing the first full 4D segmentation of the intercalating Drosophila epithelium through the use of Lattice Light Sheet Microscopy (LLSM). We find that intercalation can be initiated at any position we have surveyed along the apical-basal axis. This is striking as previous studies have largely implicated apical force generation, and a single study has suggested that contractile forces can also originate from the basal surface of the epithelium. In the proposed project, we are developing the tools to perform the first comprehensive, quantitative 3D analysis of cell intercalation in the early Drosophila embryo. We will then determine the molecular mechanisms driving 3D force generation, and whether different mechanical regimes exist across the apical-basal axis. Preliminary data suggests highly novel dynamic Myosin II and F-actin populations that show rapid axial propagation in lateral and basal regions. The 3D distributions of these populations are being mapped and the relevant actin nucleating and Myosin regulatory networks will be determined. These results will provide the first comprehensive understanding of the cortical and contractile networks that determine the mechanical environment of a gastrulating epithelium. We will also use 3D data sets in wild-type and functionally compromised backgrounds to examine how epithelial forces propagate along apical-basal and planar dimensions using topological mapping metrics. We will determine how far, and at what velocities, contractile forces spread in an intact, developing epithelium. These results will give fundamental answers into how the viscous cytoplasm and elastic cell cortex respond to force-driven displacements, and how these displacements spread within individual cells and across tissues to drive new tissue topologies.